The final objective of this proposal is to develop inhibitors of the picornaviral 3C proteinases for use in anti-picornaviral therapy. Using our recently obtained crystal structure of the hepatitis A virus (HAV) enzyme, we intend to refine our understanding of the molecular basis of proteolytic specificity and use a computer assisted approach to screen and design potent and selective inhibitors for this novel class of cysteine proteinase. As the HAV-3C proteinase structure is the only one publicly available to date, and the enzyme has been well characterized on a biochemical level, it will serve as our initial target Using the crystallographic coordinates, the closely related human rhinovirus (HRV) and poliovirus 3C proteinases, will be modeled and analogous structure- function and inhibitor development studies undertaken. To achieve this goal, a combination of computational and experimental studies are proposed to refine our current structural model, analyze the interaction of substrates and peptide-based inhibitors with the HAV3C proteinase, develop a working pharmacophore and identify potential lead compounds and second generation inhibitors. With the crystal structure now in hand, we are poised to begin "rational design" and development of small molecule inhibitors of the HAV-3C proteinase and expand our studies to other picornaviral 3C proteinases. We anticipate that insights gained from each of these systems will complement one another to accelerate and deepen our understanding of this new class of cysteine proteinase and facilitate the development of effective and highly specific inhibitors.